The efficient fractionation of biological particles from cell or tissue lysates or homogenates is crucial to the development of structural biology as a tool in applied proteomic and genomic technology. Subsequent identification and characterization of the particles recovered via fractionation may be facilitated by the method described herein. For example, the fractionation and enrichment of low abundance proteins from organelles is central to biomarker discovery in pharmaceutical drug development. Due to the heterogeneity of organellar particles, methods for isolation and characterization of functional low-abundance proteins specific to organellar compartments currently are very expensive, complex and time-consuming. Typically, standard methods for isolation of organelles involve multi-step centrifugations, including differential and density gradient ultracentrifugation, or electrophoretic separations. These methods usually require 24 to 48 hours to obtain acceptable separations. Furthermore, these classical fractionation methods have not kept pace with the increased sensitivity in protein analysis. The ability to supply a desired biological particles or organelles quickly and efficiently using the method described herein meets an unmet pharmaceutical and biomedical need for the identification of diagnostic markers in disease processes. Consequently, the need for an efficient, reproducible, and scalable method for the fractionation of biological particles has grown and will continue to proliferate as structural biology reveals new target proteins and organelles as points of possible intervention in the treatment of human disease processes.
Separation and purification of nano- and micro-particles are essential for any technical applications in numerous industrial fields including drug development, drug delivery, biosensors, coatings, and pigments. In recent research, aptamer-modified magnetic nano-particles and fluorescent nano-particles were demonstrated to have potential applications for cancer and other medical diagnostics (Herr, J. K. et al, Anal. Chem. 2006, 78(9), 2918-2924). Significant advantages have been shown in using bioconjugated nanoparticles for biosensing and bioimaging, such as cell staining, DNA detection and separation, rapid single bacterium detection, and biotechnological application in DNA protection. (Sahoo, S. K. and Labhasetwar V., Drug Discov Today. Dec. 15, 2003; (24): 1112-20) Fractionation and separation of these nano-particles to generate defined or uniform particles prior to or after applications can benefit from the method described herein.
Separation of particles can be accomplished by simple gravity sedimentation. In this procedure, the samples are allowed to sit and separation occurs due to the differences in the size and shape of the particles. Gravitational sedimentation has limited practical value for particles under a few micrometers in diameter due to the prohibitively long settling times. The sedimentation process can be accelerated by coupling driving forces including centrifugal force, magnetic force, electric force and other forces.
The most common methods to separate particles have involved the use of centrifugation including, differential, rate-zonal and isopycnic centrifugation. Overall the velocity of sedimentation of particles in a centrifugal field is described by the Stokes' equation:
  v  =                              d          2                ⁡                  (                                    ρ              p                        -                          ρ              l                                )                            18        ⁢        η              ⁢    g  
where
v=sedimentation velocity
d=diameter of particle
ρ1=density of medium
ρp=density of particle
g=gravitational field (RCF)
η=viscosity of medium
This velocity, the sedimentation velocity, is determined by the size, density and shape of the particle, as well as the viscosity of the medium through which it must travel and the centrifugal force generated.
In differential centrifugation, the suspension of particles in a medium is placed in a centrifuge tube and centrifuged for a specific period of time using a specific relative centrifugal force (RCF) to separate the group of largest particles. Then the supernatant is removed from the pellet into a separate tube, and recentrifuged for another specific period of time with another specific RCF to collect the group of the next largest particles. A series of pellets are obtained by the application of incremental increases in the RCF and time. In this process, the density of the medium is kept constant, particles are separated by changing centrifugal time and RCF, and the collected pellets are the resulting fractions of the separation. Obtaining a stable pellet using differential centrifugation depends upon the RCF, sedimentation velocity, and distance the cellular particle has to travel. The longer a sample is subjected to a specific RCF, the more likely the pellet will become contaminated by smaller particles intermixed with larger particles. The long sedimentation pathway between, and the difference in the RCF between the top and the bottom of the sample are primarily responsible for the lack of resolution and poor recoveries associated with the use of differential centrifugation. Differential centrifugation is an effective method at separation of particles into broad size classes but not suitable for separation of particles of similar sizes.
A method that results in greater resolution than differential centrifugation of all particle sizes is rate zonal gradient centrifugation. Practically, this is achieved by layering a suspension of particles on top of a preformed density gradient and then subjecting the sample to a specific RCF. Each particle size will migrate as a zone or band at a characteristic velocity. In this method, the density of the particles is always greater than the density of the liquid. In a continuous gradient, the density increases in a linear or non-linear fashion. By allowing the particles to sediment through such a density gradient, the resolution of particles is generally improved. The particles move down through the gradient in the form of discrete zones at a rate that depends primarily on their size. The centrifugation time needs to be tightly controlled—just long enough to separate the particles of interest. If the centrifugation time is too short, the particles will not separate sufficiently. If the centrifugation time is too long, some or all of the particles will end up in a pellet at the bottom of the tube. Another important limitation of using this method is that the volume of the sample is typically no more than about 5% by volume of the total volume of the density gradient used in the separation. Band broadening occurs when the capacity of the gradient is exceeded. A concentrated band of sedimentation particles can raise the density of the sample zone above that of the gradient immediately below it, leading to instability and band broadening.
Another commonly used centrifugation method for separation of particles is isopycnic centrifugation, which is also called equilibrium density gradient centrifugation. In this process, particles are either layered onto a gradient or dispensed throughout the gradient. Under centrifugal force, particles separate based on their buoyant densities, i.e., the particles migrate to a position within the gradient where their densities equal the density of the surrounding medium, called their isopycnic position. The advantage of isopycnic centrifugation over rate zonal density centrifugation is that the particles accumulate at their own equilibrium densities within the gradient as a result of the centrifugal force and the counteracting buoyant density of the gradient. Unlike rate zonal centrifugation, the sample volume may be as large as 80% of the total liquid volume. However, the resulting sample separation is distributed throughout the total fluid volume. Since it will take an infinite time to reach true equilibrium, the biggest limitation of isopycnic centrifugation is the damage to biological particles, particularly organelles, which is much greater than other methods. Because the centrifugation times are much longer, particles are exposed to potential damage or decomposition by both centrifugal force and high density gradients for extended periods of time. Additionally, long gradient columns may result in hydrostatic pressures sufficient to damage cell organelles.
Both rate zonal and isopycnic density centrifugation methods require the use of a density gradient medium for centrifugation to ensure stable sedimentation. Density gradients for isopycnic centrifugation can be either continuous, such as linear, convex, or discontinuous, while continuous density gradients are required for rate zonal centrifugation. Preparation of density gradients requires a certain level of skill in the art. Generally, density gradients can be prepared by using either a diffusion method, which requires up to 24 hours to form the gradient, or a gradient mixer. However, the shape and steepness of the gradient depends on the type of medium and the centrifugal force as well as the type of rotor used. Hence, it is difficult to ensure that the correct shape of the gradient is obtained. Another major disadvantage of both rate zonal and isopycnic density gradient centrifugation is defining the exact density of resulting fractions. Special instruments, such as refractometers, pycnometers and density meters, as well as calculations are required to determine the density of each resulting fraction.
Another commonly used method for the specific separation of lipoprotein particles is sequential flotation ultracentrifugation (Potts, J. L. et al., Clin Chim Acta, 1994, 230 (2), 215-220). In this process, the density of lipoprotein-containing liquid samples, such as plasma or serum, is adjusted by addition of solid salts, such as sodium chloride or sodium bromide, and subjected to ultracentrifugation. The top of the supernatant is removed, either by cutting off the upper portion of the centrifuge tube or by pipetting. Heavier lipoprotein particles are obtained by increasing the density of the remaining liquid sample by the further addition of solid salt, and further ultracentrifugation. Each ultracentrifugation step generally requires 20-40 hours. In addition to the disadvantage of lengthy process times, the sequential removal of substantial amounts of sample at each step and the need for the addition of solid salt significantly limits the utility of this method.
Magnetism is another force that can be used for the separation of particles. An applied magnetic field acting on micrometer and submicrometer particles having diamagnetic or paramagnetic susceptibility causes their movement. This process has been used in pigment production, nanomagnetics production for electronics and in bio-separation. Although the applied magnetic field can possibly exceed the centrifugal force, it has not been used to accelerate particle sedimentation.
Particles can also be separated by electrophoresis, a method which can separate particles based on their inherent charge and size and their subsequent migration in an applied electric field. For example, organelles are charged at neutral pH due to the presence of acidic and basic groups on their surface and will migrate in an applied electric field. The rate of movement is proportional to the charge and inversely proportional to the viscous drag, hence the rate of movement is strongly influenced by particle size. A limitation of this method is stabilizing the migrating zone of particles. Also, uneven heating of the liquid is generated by the electric current. Moreover, most of the major organelles appear to have rather similar electrophoretic mobility and it is often necessary to resort to modification of the surface charge enzymatically before a satisfactory separation can be achieved.
The instant method does not require the use of density gradients to separate particles. The separation media used are solutions of a specific density which can be incrementally adjusted during the practice of the method. The separation of particles is based on a step-wise gradient extraction of particles based on their density. The method of the invention relies upon the difference in density between subsets of particles in the sample and that of the extracting medium. Based on the Stokes' equation, particles that are equal to or lower in density than the density of the extracting medium will not pellet in the medium. Practically, these particles will float in the extracting medium while particles higher in density than the density of the medium will sediment towards the bottom of the extracting medium and form a pellet during centrifugation. The suspended particles may exist as a colloidal or polymeric mixture. Thus, the surface of the extracting medium is essentially a density barrier preventing the sedimentation of particles that are less dense than or equally as dense as the extracting medium. In this manner, the supernatant containing particles with a density less than or equal to the density of the extracting medium (ρ11) can be isolated. The remaining pellet can then be resuspended in an extracting medium where the density has been increased incrementally (ρ12) and then centrifuged at a specific speed over a specific period of time resulting in a supernatant containing particles that have a density in the range greater than (ρ11) and less than or equal to (ρ12).
The remaining pellet can then be optionally treated iteratively by the above method to obtain additional particles present in a sample depending upon their density. This iterative treatment may be optionally repeated until a desired density of the medium is reached or until no pellet is produced via centrifugation. Thus, the method demonstrates a step-wise extraction of particles from a pellet of a sample which is capable of differentiating substantially all the particles inherent to a particular sample via their density without the use of any type of density gradient currently used in most, if not all, centrifugal fractionations of particles.
The design of the centrifuge and the type rotor(s) employed to practice the above described method varies but the most common suitable design is based on a horizontally rotatable hollow disk driven by an air compressor which can be set to run at a wide variety of speeds to enable the fractionation of a wide variety of particles. Prepared samples can be loaded into the center of the horizontally rotatable hollow disk, at rest or in motion, at the start of the analysis. The rotation of the disk carries the various particles of the samples either to the surface of, or to within the suspension of, the liquid volume of the extracting medium of specific density contained within a sedimentation chamber in the horizontally rotatable hollow disk or to the pellet formed within the sedimentation chamber. After a specific period of time, the supernatant containing the extracted particles is removed by aspiration or other means from the sedimentation chamber. A new liquid volume of extracting medium of specific density then is added to resuspend the resultant pellet formed from the initial centrifugation of the sample. The method may then be repeated as many times as desired for the particular sample being analyzed.
Accordingly, there is a significant need in proteomic and genomic technology for an efficacious method for the fractionation of particles that is scale invariant, easily automated, generic for a broad range of particles and economical. It has now been surprisingly found that the use of step-wise density gradient extraction allows the defined fractionation of a wide variety of particles under conditions which are scale invariant, easily automated, and economically feasible for analytical to preparative samples.